Labeled 5,5-diphenylhydantoin derivatives for radioimmunoassay

ABSTRACT

Derivatives of 5,5-diphenylhydantoin having the following structural formula: ##STR1## where R is 2-acetyl, 3-propionyl, 4-butyryl or 5-valeryl and X is an unlabeled or radioiodinated amino radical; e.g., (tyrosine, histidine, tyrosinol, histamine). A preferred compound is radioiodinated 5,5-diphenyl-3-(5-valeryl-N-tyrosine) hydantoin. The iodine isotope may be iodine -125 or iodine-131.

This is a division of application Ser. No. 673,853, filed Apr. 5, 1976now U.S. Pat. No. 4,092,479.

This invention relates to the radioimmunoassay of the anti-epilepticdrug, 5,5-diphenylhydantoin and pertains more specifically to newchemical compounds which are derivatives of 5,5-diphenylhydantoin andwhich, in radioiodinated form, can be used as tracers, and to the methodof making them.

Cook et al. in Research Communications in Chemical Pathology andPharmacology, Vol. 5, No. 3, p. 767 (1973) have reported the use of5,5-phenylhydantoin-3-(5-valeric acid) conjugated to bovine serumalbumin as an antigen to elicit an antibody response in rabbits for theradioimmunoassay of 5,5-diphenylhydantoin. However, the tracer used was5,5-bis-(phenyl-4-³ H)-hydantoin necessitating scintillation counting ofthe tritium isotope, which is less convenient than gamma counting ofiodine isotopes. Tritium labeled tracers also generally have a lowerspecific activity than iodinated tracers, which may limit thesensitivity of the radioimmunoassay.

An object of this invention is to provide new derivatives of5,5-diphenylhydantoin which are useful in the preparation of tracercompounds for the radioimmunoassay of 5,5-diphenylhydantoin as well asto provide the tracer compounds themselves. This is accomplished bymaking derivatives with the following structural formula: ##STR2##

In the foregoing structural formula, ##STR3## is a straight or branchedchain aliphatic acyl group in which R contains from 1 to 7 carbon atomsand in which R may contain, in addition to carbon and hydrogen, up totwo hydroxyl and amino groups. Among the acyl groups included are2-acetyl, 3-propionyl, 4-(2-hydroxybutyryl), 4-(3-hydroxybutyryl),3-(2-methylpropionyl), 5-valeryl, 4-isovaleryl, 4-(3-aminoisovaleryl),4-(2-hydroxyisovaleryl), 6-caproyl, 6-(2-aminocaproyl), 7-enanthyl,8-caprylyl, 6-(2-ethylcaproyl), 8-(2-hydroxycaprylyl); of these,5-valeryl is preferred.

The moiety identified as X in the foregoing structural formula isselected from the group consisting of the following ring-containingamino radicals: ##STR4## in which Y is hydrogen, fluoro, or lower alkyl(up to 7 carbon atoms), preferably methyl; and amino-terminatedpolypeptide radicals containing one or more of the aforesaidring-containing radicals. The ring-containing radicals may be unlabeledor radiolabeled, preferably radioiodinated with one radioiodine atomwith the probable positions of labeling indicated by an asterisk. When Yis hydrogen, the ring-containing radicals may be radioiodinated with tworadioiodine atoms, one being in the Y position, but this is notpreferred. The radioiodine is generally ¹²⁵ I, but other iodine isotopessuch as ¹³¹ I may also be used. Radiolabeling is preferably carried outafter coupling of the ring-containing radical is complete.

These compounds are prepared from the 3-sodium salt of5,5-diphenylhydantoin, which is reacted with the ω-halogenated loweralkyl ester of the desired carboxylic acid to produce5,5-diphenylhydantoin-3-(ω-carboxylic acid ester). The ester is thenconverted to the corresponding acid by hydrolysis. The5,5-diphenylhydantoin-3-(ω-carboxylic acid) is then coupled to thedesired ring-containing amino compound via carbodiimide, or ethylchloroformate activation of the carboxyl group. The resulting compoundis then iodinated, preferably by the chloramine T method.

The precursors of compounds of the present invention are prepared byfirst reacting the sodium salt of 5,5-diphenylhydantoin withω-halogenated carboxylic acid lower alkyl ester in an inert solvent asdescribed by Cook et al. While Cook et al. employed methyl5-bromovalerate as the ω-halogenated carboxylic acid lower alkyl ester,other lower alkyl esters of ω-halo aliphatic carboxylic acids having thestructure ##STR5## in which R is defined as above may also be used toprovide the desired aliphatic acyl group. ω-Bromocarboxylic acid loweralkyl esters are the preferred halo-derivatives, but chloro- andiodo-derivatives may also be employed. The alkyl portion of the estermay contain one to seven carbon atoms or even more although the methyland ethyl esters are preferred. The5,5-diphenylhydantoin-3-(ω-carboxylic acid ester) is then hydrolyzed asdescribed by Cook et al. to form the corresponding free acid.

The 5,5-diphenylhydantoin-3-(ω-carboxylic acid) is then coupled to thedesired ring-containing amino compound using either ethyl chloroformate,isobutyl chloroformate or pivaloyl chloride to generate mixedanhydrides. This reaction is carried out in any conventional aproticsolvent at low temperature (0°-10° C. ) under anhydrous conditions.Dioxane is the preferred solvent and one equivalent of organic base suchas triethylamine, etc., is added to consume hydrochloric acid generatedin the mixed anhydride formation. The ring-containing amino compound tobe coupled to the mixed anhydride is added to the reaction mixture ineither aqueous solution, or in a mixed organic solvent; the reaction ofthe amino compound with the anhydride is apparently more rapid thanhydrolysis of the anhydride.

Isolation of the desired compound is accomplished by addition of thereaction mixture to acidified water. The precipitate obtained can berecrystallized from ethanol. Further purification may be accomplished bypreparative thin layer chromatography, gel filtration, affinitychromatography, or other suitable procedures.

The radioiodinated derivatives may be prepared by any one of thefollowing methods:

(1) Chloramine T Method of Hunter-Greenwood, W. Hunter, R. C. Greenwood,Nature, 194, 495 (1962);

(2) Iodine Monochloride Method, M. Ceska, F. Grossmuller, U. Lundkvist,Acta Endcrinologia, 64, 111-125 (1970);

(3) Isotopic Exchange Method, R. E. Counsell, V. V. Ranade, P. Pocha, R.E. Willette, W. Diguilio, J. Pharmaceut. Sciences, 57, 1657 (1968);

(4) Electrolytic Iodination, R. Pennisi, U. Rosa, J. Nuclear Biol. andMedicine, 13, 64 (1964); and

(5) Enzymatic Iodination, H. Van Vanakis, J. J. Langone, L. J. Riceberg,L. Levine, Cancer Research 34, 2546-2552 (1974).

The marginally water soluble products of this invention may be iodinatedin inert solvents such as water or water-alcohol mixtures.

Separation of unreacted radioactive iodine is accomplished by gelfiltration and the use of aqueous solvents that elute selectivelyunreacted inorganic iodide and the desired iodinated product.

EXAMPLE 1

A. A mixture of 1.1 gm of 3-sodium 5,5-diphenylhydantoin and 0.8 gm ofmethyl 5-bromovalerate in 20 ml of dimethylformamide is heated withstirring to 60° C. for 3 hours. The reaction mixture is added to 300 mlof 30% saturated aqueous ammonium sulfate and allowed to crystallize for72 hours to yield crude 5,5-diphenylhydantoin 3-(5-valeric acid methylester), m.p. 84°-90° C. On recrystallization from methanol 0.67 gm of awhite crystalline material is obtained, m.p. 94°-96° C.

B. The ester obtained in 1-A was hydrolyzed by refluxing for 3 hours in0.5 normal hydrochloric acid in 10% aqueous dioxane. The desired acidcrystallized on standing at 4° C. for 18 hours. The crude acid had am.p. of 135°-145° C. which was raised to 161°-163° C. onrecrystallization from ethyl acetate. A yield of 280 mg of the pure acidwas obtained starting with 0.67 gm of ester.

C. Fifteen mg of 5,5-diphenylhydantoin 3-(5-valeric acid) was added to250 microl. of dioxane in a 12 × 75 mm glass test tube. The acid wentinto solution on the addition of 50 microl. of 10% triethylamine indioxane. After chilling to about 10° C. in an ice water bath, 50 microl.of 10% ethyl chloroformate in dioxane was added and allowed to react for30 minutes at 10° C. to form the mixed anhydride.

L-Tyrosine, 25.1 mg, was dissolved in 0.5 ml distilled water to which 5drops of triethylamine had been added. The aqueous L-Tyrosine was addedto the cold mixed anhydride solution and allowed to react for 2 hourswhile the reaction mixture slowly came to room temperature. The entirereaction mixture was added to 5 ml of distilled water acidified with 5drops of concentrated sulfuric acid and allowed to precipitate for 18hours at 4° C.

The precipitate was collected by vacuum filtration and recrystallizedfrom ethanol yielding 3 mg of product:5,5-diphenylhydantoin-3-(5-valeryl-N-tyrosine).

Analogous products of the present invention can be made by substitutingother ω-halocarboxylic acid lower alkyl esters for methyl5-bromovalerate and by substituting, in place of tyrosine, otherring-containing amino compounds such as tyrosinol, 4-(2-aminoethyl)phenol, histidine, histidinol, histamine, or aminoterminated polypeptidecontaining one or more of the X radicals as defined above.

EXAMPLE 2

Radioiodination of 5,5-diphenylhydantoin-3-(5valeryl-N-tyrosine) waseffected by the method of Hunter and Greenwood. 10 microl. of a solution(1 mg in 7.0 ml of ethanol) of5,5-diphenylhydantoin-3-(5-valeryl-N-tyrosine) was diluted in a clean 12× 75 mm glass test tube with 120 microl. of 0.5 molar phosphate buffer,pH 7.4. To this mixture was added 1mCi of sodium iodide ¹²⁵ I in 20microl. of 0.1 normal NaOH, followed by 20 microl. of an aqueoussolution of chloramine T (70 mg in 10 ml of distilled water).

After reaction at room temperature for 2 minutes with occasional shakingthe reaction was quenched by the addition of 40 microl. of a solution ofsodium metabisulfite (70 mg in 10 ml of distilled water). The reactionmixture was transferred with a Pasteur pipet to a 1 × 10 cm SephadexG-10 column that had previously been equilibrated with a 0.1 molarphosphate buffer, pH 8.5. The equilibration buffer was used to eluteunreacted sodium iodide. Five ml fractions were collected.

About one-third of the radioactivity was eluted in the phosphate buffer.The remaining activity was retained on the column until the elutingbuffer was changed to distilled water. Four 5 ml water fractions werecollected; these contained about two-thirds of the radioactivityinitially put on the column and represented the desired products:5,5-diphenylhydantoin-3-(5-valeryl-N-3-¹²⁵ I tyrosine) and5,5-diphenylhydantoin-3-(5-valeryl-N-(3,5-¹²⁵ I) tyrosine).

The radioiodinated derivatives of the present invention may be used asthe labeled antigen in the radioimmunological assay of5,5-diphenylhydantoin. A radioimmunological procedure which may be usedis a modification of the one employed by R. E. Tigelaar, R. L. RapportII, J. K. Inman and H. J. Kupferberg, Clinica Chimica Acta 43, 231-241(1973) for carbon-14 labeled 5,5-diphenylhydantoin.

To 1 ml of pH 7.4 0.01 molar tris buffer which is isotonic in saline andcontains 0.1% bacitracin is added 50 microl. of serum, which had beendiluted 1 to 100 in distilled water in a 12 × 75 mm glass test tube.About 40 picograms of the radioiodinated derivative described herein,containing about 30,000 counts per minute is added as well as sufficientanti-5,5-diphenylhydantoin rabbit antibody to produce 30 to 60 percentbinding in the absence of unlabeled drug.

The mixture is incubated for one hour at 25° C. Competition for alimited number of antibody binding sites by the labeled compound and thedrug determines the amount of antibody-bound tracer at equilibrium.Separation of bound and free radioiodinated compound is achieved by thedextran-coated charcoal method of Herbert et al. J. Clin. Endocr. 25,1375-1384 (1965), resulting in selective removal of free labeled andunlabeled compound by the charcoal, which is then separated bycentrifugation and decanting. The supernatant containing the antibodybound labeled and unlabeled drug is counted in a gamma counter.

The radioiodinated derivatives of the present invention, unlike the ¹⁴ Cand ³ H tracers employed by Tigelaar et al. and Cook et al.respectively, may be used with a plastic tube which has been coated witha specific antibody to 5,5-diphenylhydantoin in a procedure similar tothat described by Catt et al., Science 158, 1570 (1967).

Patient serum to be assayed is diluted 1 part to 100 parts of distilledwater. An aliquot of 50 microl. of the diluted serum is introduced intothe bottom of the antibody coated tube. One ml of a pH 7.4 buffercontaining 0.01 molar tris, 0.15 molar NaCl, 0.1% bacitracin and 30 pgor 30,000 cpm of the radioiodinated derivative of the present inventionis added to the diluted patient sample in the antibody coated tube. Thereaction mixture is shaken briefly and allowed to incubate at 37° C. for45 minutes. Competition between the unlabeled drug in the patient serumand the labeled derivative for the limited number of antibody bindingsites available on the side of the tube determine the amount of antibodybound tracer, when the binding reaction is terminated. Separation ofantibody bound and free tracer is effected by aspirating or decantingthe aqueous phase that contains the free tracer. The tube containing theimmobilized antibody bound tracer is counted in a gamma counter.

The compounds of the present invention allow the detection andquantitation of serum levels of 5,5-diphenylhydantoin with greatersensitivity and ease than had been possible previously with tritiated orcarbon-14 labeled tracers, which suffer from relatively low specificactivity and the necessity for the more laborious and cumbersomescintillation counting relative to gamma counting.

What is claimed is:
 1. The method of assaying the 5,5-diphenylhydantoin content of serum which comprises bringing the serum into contact with (1) a radioiodinated derivative of a compound selected from the group consisting of compounds having the following structural formula: ##STR6## wherein R is selected from the group consisting of straight and branched aliphatic acyl groups in which R contains from 1 to 7 carbon atoms and in which R may contain, in addition to carbon and hydrogen, up to two hydroxyl and amino groups, and wherein X is selected from the group consisting of the following ring-containing amino radicals: ##STR7## wherein Y is hydrogen, fluoro, or lower alkyl, and with (2) 5,5-diphenylhydantoin antibody in a buffer to form an assay mixture,incubating said assay mixture to permit said radioiodinated compound and the 5,5-diphenylhydantoin in the serum to bind competitively to the antibody, separating the assay mixture into two portions, one containing antibody-bound radioiodinated compound and the other containing free radioiodinated compound, and measuring the radioactivity of one of said portions.
 2. The method as claimed in claim 1 in which the compound is radioiodinated 5,5-diphenylhydantoin-3-(5-valeryl-N-tyrosine). 